THE  UNIVERSITY 

OF  ILLINOIS 

LIBRARY 


wo.  I 


1 


UNIVERSITY  OF  ILLINOIS 

Agricultural  'Experiment  Station 


BULLETIN  No.  190 


SOIL  BACTERIA  AND  PHOSPHATES 


BY  CYRIL  G.  HOPKINS  AND  ALBERT  L.  WHITIN© 


URBANA,  ILLINOIS,  JUNE,  1916 


The  fundamental  facts  of.  soil  fertility  and  crop  production  are 
slowly  but  surely  becoming  established,  and  the  farmer  has  begun  to 
shape  his  agricultural  practice  by  the  discoveries  of  science.  Of 
these  discoveries  the  following  may  fairly  be  called  fundamental. 

The  fact  established  by  Senebier  of  Switzerland  that  the  car- 
bon of  crops  is  derived  from  the  atmosphere  and  not  from  the  soil. 

The  work  of  DeSaussure  which  clearly  demonstrated  that  min- 
eral elements  are  essential  to  plant  growth. 

The  work  of  Lawes  and  Gilbert  showing  that  farm  crops  are 
unable  to  take  the  nitrogen  from  the  atmosphere  thru  the  leaf. 

The  experiments  of  Atwater  and  Woods  establishing  the  fact 
that  leguminous  crops  are  in  some  way  able  to  utilize  atmospheric 
nitrogen. 

The  experiments  of  Hellriegel  establishing  the  fact  that  the 
principal  agents  in  the  fixation  of  atmospheric  nitrogen  are  cer- 
tain bacteria  growing  upon  the  roots  of  leguminous  plants. 

Investigations  at  the  University  of  Illinois  concerning  the 
amounts  of  nitrogen  which  may  be  brought  into  the  soil  by  means 
of  these  bacteria,  under  field  conditions.1 

Later  experiments  by  the  University  of  Illinois  concerning  the 
availability  of  natural  sources  of  mineral  plant  food  in  permanent 
agriculture.1 

The  purpose  of  the  experiment  herein  reported  was  to  determine 
whether  the  farmer  supplying  nitrogen  to  his  soil  in  the  form  of  or- 
ganic matter  could  depend  upon  its  decomposition  to  render  soluble 
the  finely  ground  rock  phosphate,  and  so  maintain  the  necessary  sup- 
ply of  available  phosphorus.  This  probability  has  long  been  suspected 
by  the  University,  and  the  results  reported  seem  to  indicate  conclu- 
sively that  in  the  presence  of  liberal  applications  of  finely  ground  rock 
phosphate  such  solution  may  be  depended  upon.  This  discovery  ranks 
in  importance  with  the  inoculation  of  legumes  and  with  the  deter- 
mination of  the  amount  of  atmospheric  nitrogen  that  can.be  fixed  by 
leguminous  crops. 

When  we  remember  that  chemistry  as  a  science  dates  practically 
from  the  time  of  the  American  Revolution,  when  King  George  was 
more  interested  in  better  agriculture  for  England  than  in  quieting  his 
turbulent  colonies,  and  when  we  recall  that  the  great  science  of  bac- 
teriology has  entirely  developed  since  the  time  of  our  Civil  War  and 
the  abolition  of  slavery,  it  is  evident  that  rapid  progress  is  being  made 
in  the  establishment  of  farming  on  a  truly  scientific  basis,  and  that 
slow  as  discovery  seems  to  be,  it  is  after  all  relatively  rapid. 

E.  DAVENPORT,  Director. 

'See  HI.  Agr.  Exp.  Sta.  Buls.  76  (Alfalfa  on  Illinois  Soil),  94  (Nitrogen 
Bacteria  and  Legumes),  182  (Potassium  from  the  Soil). 


SUMMARY  OF  BULLETIN  No.  190 

1.  Nitrite   bacteria   make   phosphorus   and   calcium   soluble   from   insoluble 
phosphates  when  they  oxidize  or  convert  ammonia  into  nitrite.  Page  401 

2.  The  actual  ratio  found  shows  that  about  one  pound  of  phosphorus  and 
about  two  pounds  of  calcium  are  made  soluble  for  each  pound  of  nitrogen  oxidized, 
aside  from  the  action  of  the  acid  radicles  associated  with  the  ammonia. 

Page  402 

3.  The  ratio  of  solubility  found  on  the  basis  of  nitrogen  to  phosphorus  and 
calcium  conforms  to  the  following  reaction: 

4HNO3+Ca3(PO4)2^CaH4(P04)2+2Ca(NO2)a 

According  to  this  equation,  56  pounds  of  nitrogen  liberate  in  soluble  form  62 
pounds  of  phosphorus  and  120  pounds  of  calcium.  Page  403 

4.  Plants  are  important  factors  in  the  liberation  of  phosphorus,  owing  to  the 
production  of  carbon  dioxid  and  the  removal  of  the  soluble  phosphorus  produced 
by  the  bacteria.  Page  404 

5.  Neither  ammonia-producing  bacteria  nor  nitrate  bacteria  liberate  appre- 
ciable amounts  of  soluble  phosphorus  from  insoluble  phosphates. 

Pages  398,  403 

6.  Other  acid-producing  bacteria  make   phosphorus   soluble  from  insoluble 
phosphates  according  to  the  nature  and  amount  of  the  acid  produced. 

Pages  403,  405 

7.  A  comparison  of  the  amounts  of  nitrogen,  phosphorus,  and  calcium  re- 
quired by  farm  crops,  with  those  possible  of  solution  by  biochemical  action,  shows 
possibilities  far  beyond  the  plant  requirements;  which  leads  to  the  conclusion  that 
plenty  of  rock  phosphate  in  contact  with  decaying  organic  matter  must  give  the 
plants  an  excellent  opportunity  to  obtain  both  phosphorus  and  calcium  as  well  as 
nitrogen.  Page  405 


SOIL  BACTERIA  AND  PHOSPHATES 

BY  CYRIL  G.  HOPKINS,  CHIEF  IN  AGRONOMY  AND  CHEMISTRY,  AND 
ALBERT  L.  WHITING,  ASSOCIATE  IN  SOIL  BIOLOGY 

Raw  rock  phosphate  is  by  far  the  cheapest  source  of  phos- 
phorus to  apply  to  soils.  It  consists  chiefly  of  tricalcium  phosphate, 
Ca3(P04)2,  which  is  the  most  common  form  of  phosphorus  in  the  great 
natural  deposits.  This  phosphorus  compound  is  relatively  insoluble  in 
water,  and,  for  this  reason,  it  has  been  argued  by  some  that  it  does  not 
become  available  to  plants ;  but  long-continued  field  experiments,  pot- 
culture  experiments,  and  farm  practice  have  fully  demonstrated  that 
this  kind  of  phosphate  does  become  available  for  plant  growth.  (See 
Circulars  181  and  186). 

With  the  results  obtained  in  practice  confirming  on  a  large  scale  the 
experimental  results,  it  became  a  laboratory  problem  to  determine  how 
this  insoluble  phosphate  is  made  soluble  by  changes  occurring  in  soils. 
The  increased  beneficial  results  obtained  by  following  the  practice  com- 
monly recommended  of  intimately  mixing  decaying  organic  matter 
Vith  the  phosphate  lead  to  the  suggestion  that  the  action  of  the  soil 
bacteria  that  decompose  organic  matter  might  be  an  important  factor 
in  the  solution  of  the  phosphate.  The  investigation  reported  in  this  bul- 
letin has  proved  that  this  hypothesis  was  correct,  for  these  bacteria 
have  been  found  capable  of  making  the  phosphate  soluble.  The  data 
hereinafter  recorded  show  that  the  action  is  even  more  direct  than  we 
had  reason  to  believe.  It  has  been  the  common  teaching  that  nitrifying 
bacteria  require  the  presence  of  a  free  base,  such  as  lime  or  an  alkaline 
carbonate,  but  we  have  found  that  the  bacterial  action  produces  acid 
phosphate  and  proceeds  in  the  presence  of  this  acid  salt. 

DECOMPOSITION  OF  ORGANIC  MATTER  BY  SOIL  BACTERIA 

The  organic  matter  of  soils  consists  of  the  remains  of  plant  roots, 
stems,  and  leaves,  and  of  farm  manures.  These  are  made  up  of  pro- 
teins, sugars,  starches,  fiber,  and  other  less  important  compounds,  such 
as  fats.  All  these  constituents  are  subject  to  decay  by  bacteria.  They 
are  broken  down  by  bacteria  into  less  complex  substances.  This  break- 
ing down  is  not  brought  about  by  one  kind  of  bacteria  only,  nor  is  it 
the  result  of  but  one  process. 

The  importance  of  the  action  of  decomposition  products  of  the 
active  organic  matter1  of  the  soil  on  the  solubility  of  phosphates  is 
better  understood  by  a  consideration  of  three  important  and  definitely 


MDrganic  matter  which  is  capable  of  rapid  decomposition  is  called  active  or- 
ganic matter,  and  that  which  is  resistant  to  decay  is  known  as  humus  or  inactive 
organic  matter. 

395 


396  BULLETIN  No.  190  [June, 

recognized  processes  that  have  long  been  known  to  bring  about  the 
change  of  the  nitrogen  from  the  unavailable  form,  as  it  occurs  in  the 
protein  of  clover,  manure,  etc.,  to  the  readily  available  form  of  the 
nitrate,  as  found  in  calcium  nitrate  (''nitrate  of  lime"),  sodium  ni- 
trate ("nitrate  of  soda"),  potassium  nitrate  ("saltpeter"),  etc. 

There  are  three  well  known  steps,  or  stages,  in  the  biochemical  pro- 
cess of  converting  organic  nitrogen  into  nitrate  nitrogen: 

(1)  Ammonia  Production. — The  first  process  results  in  the  change 
of  the  organic  nitrogen  to  ammonia  nitrogen.  The  ammonia  (NH3) 
is  absorbed  by  the  soil  moisture  (H20)  and  forms  ammonium  hydroxid 
(NH4OH),  or  household  ammonia.  Much  carbon  dioxid  (C02)  is  pro- 
duced-at  the  same  time,  and  some  of  it,  also,  is  absorbed  by  the  soil 
moisture  and  then  unites  with  the  ammonia  to  form  ammonium  car- 
bonate. The  formation  of  this  ammonium  carbonate  is  represented 
by  the  following  equations:1 

NH,+H20=lSni4OH   (ammonium  hydroxid) 
COj-fHjO^HjCO,  (carbonic  acid) 
2NH1OH-fH2COs=:2HaO+(NH1)jCO,  (ammonium  carbonate) 

Ammonia,  ammonium  hydroxid,  and  ammonium  carbonate  are 
alkaline.  Red  litmus  is  turned  blue  by  these  compounds.  This  should 
be  carefully  noted,  as  only  acids  are  able  to  make  phosphates  and  lime- 
stones soluble,  as  explained  elsewhere. 

If  more  carbon  dioxid  is  produced  than  will  unite  with  the  am- 
monia produced,  it  will  dissolve  in  the  soil  moisture,  forming  car- 
bonic acid,  and  it  may  make  raw  rock  phosphate  soluble,  as  will  be 
shown  later.  The  soil  moisture,  however,  will  hold  only  a  certain 
amount  of  this  gas  and  any  above  that  amount  is  returned  to  the  air, 
which  is  the  source  of  carbon  for  plants. 

While  the  ammonia  is  being  produced,  other  compounds  are  like- 
wise being  formed,  and  such  organic  acids  as  the  acetic  acid  of  vinegar, 

lpniese  equations  are  easily  understood  by  reference  to  the  following  table  of 
atomic  weights  and  valences,  nydrogen  being  taken  as  the  standard,  or  unit,  of 
measure : 

Element                             Symbol                 Atomic  weight  Valence 

Hydrogen                               H                                     1  1 

Oxygen                                     O                                     16  2 

Nitrogen                                   N                                    14  3   or  5 

Carbon                                      C                                    12  4 

Phosphorus                            P                                   31  3  or  5 

Calcium                                  Ca                                40  2 

Sulfur                                     8                                   32  2 

The  symbol  O  stands  for  an  atom  of  oxygen  with  a  weight  of  16  and  a  valence 
of  2.  The  term  valence  means  the  number  of  bonds  of  attraction  possessed  by  an 
atom.  These  bonds  may  be  considered  as  hands  or  hooks.  Thus,  the  two-handed 
oxygen  atom  may  hold  two  one-handed  hydrogen  atoms  in  the  compound  called 
water,  H2O,  or  H-O-H.  While  the  elements  hydrogen  and  oxygen  are  both  gases, 
H2O  is  the  formula  of  a  molecule  of  the  liquid  compound,  water,  whose  molecular 
weight  is  V,  the  sum  of  the  atomic  weights. 

2083(1*04)2  is  read,  "two  Ca  three,  PO  four  twice,"  and  means  two  mole- 
cules of  tricalcium  phosphate,  each  consisting  of  three  atoms  of  calcium  and 
twice  the  parenthetic  group  containing  one  atom  of  phosphorus  and  four  of 
oxygen.  One  molecule  weighs  310  (times  the  hydrogen  atom),  as  readily  com- 
puted from  the  atomic  weights. 


1916]  SOIL  BACTERIA  AND  PHOSPHATES  397 

the  lactic  acid  of  sour  milk,  the  butyric  acid  of  rancid  butter,  and 
many  others  result.  These  will  unite  with  bases,  such  as  lime,  or  will 
react  with  and  decompose  such  mineral  compounds  'as  raw  rock  phos- 
phate and  ground  limestone.  In  this  way  they  will  dissolve,  or  lib- 
erate, phosphorus  and  calcium. 

The  potato  and  the  hay  bacteria,1  which  are  very  common  in  all 
soils,  are  vigorous  producers  of  ammonia.  Many  other  kinds  of  soil 
bacteria  decompose  organic  matter  into  ammonia,  carbon  dioxid,  and 
organic  acids.  These  ammonifying  bacteria,  however,  are  unable  to 
convert  ammonia  into  any  other  compounds.  By  their  action  am- 
monia is  always  being  produced  in  soils.  It  will  be  produced  rapidly 
or  slowly  according  to  the  conditions  which  prevail  in  a  given  por- 
tion of  the  soil.  The  production  of  ammonia  in  soils  seldom  ap- 
proaches that  observed  in  manure  piles  or  composts.  Small  amounts 
of  ammonia  are  always  present  in  soils  in  a  good  state  of  fertility,  but 
nitrate,  which  is  derived  from  ammonia,  is  normally  present  in  larger 
amounts.  The  presence  of  nitrate  demonstrates  that  a  change  from  an 
alkaline  condition  to  an  acid  condition  is  always  taking  place,  for  to 
produce  nitrate  requires  nitrous  or  nitric  acid.  Even  when  manure 
and  raw  rock  phosphate  are  composted,  nitrate  forms  in  large  amounts, 
altho  a  test  of  the  mass  as  a  whole  shows  it  to  be  alkaline:  at  many 
local  points  acid  nitrogen  must  be  formed,  otherwise  nitrate  produc- 
tion would  be  impossible. 

As  ammonia  is  the  most  important  compound  obtained  in  the  first 
stage  of  the  decomposition  of  organic  matter,  this  stage  is  called  am- 
monification  or  ammonia  production. 

(2)  Nitrite  Production. — The  second  and  most  important  of  the. 
three  stages  consists  of  the  oxidation  of  the  ammonia  to  nitrite.  In 
order  to  form  a  nitrite,  nitrous  acid  (HN02)  must  be  produced.  This 
nitrous  acid  is  very  similar  to  nitric  acid  (HN03).  The  oxidation  of 
ammonia  to  nitrous  acid  by  the  nitrite  bacteria  is  represented  by  the 
following  equation : 

(NH<)  zC03+60=2HN02+HaCOs-|-2HaO 

The  ammonium  portion  of  the  ammonium  carbonate,  an  alkaline 
compound,  has  been  converted  into  nitrous  acid  and  carbonic  acid  has 
been  set  free.  Both  these  acids  will  combine  with  some  base.  It  is  im- 
portant to  note  that  nitrogen  of  the  alkaline  substance,  ammonia,  has 
been  converted,  or  transformed,  by  the  biochemical  removal  of  hydro^ 
gen  and  addition  of  oxygen  into  a  strongly  acid  substance,  nitrous  acid. 

This  strong  acid  must  be  neutralized  by  some  base,  if  nitrification 
is  to  proceed,  for  the  bacteria  are  inactive  in  the  presence  of  any 
noticeable  amounts  of  strong  acid.  If  the  acid  is  neutralized,  a  ni- 

JThe  potato  bacteria  (Bacillus  mesentericus)  are  so  called  on  account  of  their 
being  easily  found  in  the  eyes  of  potatoes;  the  so-called  hay  bacteria  (Bacillus 
subtilis  are  found  very  abundantly  in  hay  and  at  one  time  were  thought  to  cause 
hay  fever. 


398  BULLETIN  No.  190  [June, 

trite  is  the  product  formed,  such  as  calcium  nitrite,  Ca(N02)2.  The 
primary  purpose  of  this  investigation  is  expressed  in  the  question, 
Will  the  calcium  of  pure  rock  phosphate,  Ca3(P04)2,  suffice  as  a  base; 
and  if  so,  will  the  phosphorus  be  made  soluble?  This  will  be  an- 
swered by  the  experimental  data  reported  in  another  part  of  this 
bulletin. 

If  nitrite  production  takes  place  with  tricalcium  phosphate  as  a 
source  of  the  base  calcium,  then  the  reaction  must  be  represented  by 
one  of  the  following  equations: 

Ca3(PO4)2+2HN02=Ca2H2(P04)2-fCa(:NTO2)2 

or,  Ca3(PO1)2+4HNO2=CaH1(PO4).!-f2Ca(N02), 

The  bacteria  which  oxidize  the  ammonia  to  nitrite  are  called  ni- 
trite bacteria,  or  Nitrosomonas,  and  only  one  kind  is  known  which  is 
able  to  perform  this  intermediate  step  in  the  transformation  of  organic 
nitrogen  to  nitrate.  These  bacteria  are  unable  to  use  any  other  than 
ammonium  compounds. 

(3)  Nitrate  Production. — The  third  and  last  stage  is  a  simple 
oxidation  of  the  nitrite  to  nitrate  by  the  action  of  nitrate  bacteria 
(Nitrobacter) .  It  consists  in  the  addition,  by  biochemical  action,  of 
oxygen  to  the  nitrite : 

Ca(NOa)a+20=Ca(NO,)a 

This  reaction  increases  neither  acidity  nor  alkalinity,  but  it  pro- 
duces nitrate,  a  compound  of  nitrogen  which  is  preferred  by  nearly 
all  forms  of  plant  life.  No  liberation  of  insoluble  compounds  would 
be  expected  in  this  process,  as  no  additional  base  is  necessary,  as  seen 
by  reference  to  the  equation. 

These  nitrate  bacteria  live  only  on  nitrite,  and  consequently  they 
must  await  the  action  of  the  nitrite  bacteria.  While  it  is  known  that 
nitrites  are  formed  in  soils  under  field  conditions,  only  ammonia  and 
nitrate  can  be  found,  as  the  nitrate  bacteria  change  the  nitrite  as 
rapidly  as  it  is  formed. 

INFLUENCE  OF  AMMONIA  PRODUCTION  ON  SOLUBILITY 

OF  PHOSPHATES 

As  already  stated,  the  most  important  product  formed  in  the  first 
process,  or  stage,  of  the  decomposition  of  the  organic  matter  is  am- 
monium carbonate.  The  ammonium  carbonate  is  alkaline,  and  conse- 
quently could  not  be  expected  to  exert  any  action  on  the  solubility  of 
raw  rock  phosphate. 

In  1904  Stalstrom  of  Finland  conducted  laboratory  experiments  on 
the  solubility  of  pure  rock  phosphate  with  bacteria  which  produced 
ammonium  carbonate  from  peat  and  from  manure  containing  peat 
litter.  He  concluded  that  there  was  no  appreciable  increase  in  solu- 
bility of  phosphorus  where  the  bacteria  had  produced  ammonium  car- 
bonate over  the  sterile  treatments  in  which  no  ammonium  carbonate 


1916]  SOIL  BACTERIA  AND  PHOSPHATES  399 

was  produced.  His  experiments  lasted  forty-two  days  and  were  un- 
der conditions  which  would  permit  of  determining  soluble  phosphorus 
were  it  present.  His  work  is  extremely  interesting  as  it  demonstrates 
that  in  the  first  stage  of  decomposition  it  has  been  impossible  to  meas- 
ure any  soluble  phosphorus  without  the  growing  plant  as  an  indicator. 
Similar  results  have  been  obtained  by  the  Rhode  Island  and  Wis- 
consin Experiment  Stations  in  attempts  to  detect  soluble  phosphorus 
in  fermenting  mixtures  of  manure  and  raw  rock  phosphate  and  in 
mixtures  of  soil  and  raw  rock  phosphate.  When,  however,  plants  have 
been  grown  in  the  mixtures,  crop  yields  have  demonstrated  an  ad" 
vantage  from  the  intimate  contact  'of  the  phosphate  with  the  decaying 
organic  matter.  Even  when  soluble  phosphates  are  applied  to  similar 
mixtures,  it  soon  becomes  difficult  to  find  soluble  phosphorus  owing  to 
the  alkaline  condition  of  the  mass  as  a  whole.  However,  this  does  not 
preclude  the  possibility  of  phosphorus  having  been  made  soluble  at 
many  local  points,  strong  evidence  of  which  is  afforded  by  the  growing 
plants. 

SOLUTION  OF  PHOSPHATES  BY  ACTION  OF  NITRITE 

BACTERIA 

Until  the  work  reported  in  this  bulletin  was  undertaken,  the  part 
played  by  the  nitrite  bacteria  in  dissolving  mineral  compounds,  and 
particularly  raw  rock  phosphate,  had  never  been  investigated,  and  to 
determine  this  was  our  principal  object  in  these  experiments. 

One  of  the  authors  made  the  following  suggestion  several  years  ago  : 

"In  the  conversion  of  sufficient  organic  nitrogen  into  nitrate  nitro- 
gen for  a  hundred-bushel  crop  of  corn,  the  nitric  acid,  if  formed,  would 
be  alone  sufficient  to  convert  seven  times  as  much  insoluble  tricalcium 
phosphate  into  soluble  monocalcium1  phosphate  as  would  be  required 
to  supply  the  phosphorus  for  the  same  crop."2 

The  following  equation  shows  that  nitrous  acid  makes  raw  rock 
phosphate  soluble. 


Expressed  in  other  terms  :  188  pounds  of  nitrous  acid  mixed  with 
310  pounds  of  pure  rock  phosphate  make  234  pounds  of  acid  phos- 
phate and  264  pounds  of  calcium  nitrite,  both  of  which  are  soluble. 
Thus,  56  pounds  of  nitrogen,  when  oxidized  to  nitrous  acid,  have  power 
to  dissolve  62  pounds  of  phosphorus  and  120  pounds  of  calcium,  con- 
tained in  rock  phosphate.  If  the  nitrite  bacteria  bring  about  the  re- 
action shown  by  the  equation,  both  calcium  and  phosphorus  are  thus 
dissolved  from  raw  rock  phosphate.  The  bacteria  need  the  calcium  to 
neutralize  the  nitrous  acid  produced,  and  a  small  amount  of  both  cal- 
cium and  phosphorus  is  needed  for  their  own  bodies. 

^-Mono-  means  one;  di-,  two;  tri-t  three;  tetra-,  four;  pentar-,  five.  Mono- 
calcium  phosphate,  which  may  also  be  called  monocalcium  tetrahydrogen  phos- 
phate, is  an  acid  phosphate,  the  acidity  being  due  to  the  hydrogen. 

"Hopkins  '  '  '  Soil  Fertility  and  Permanent  Agriculture,  '  '  page  197. 


400  BULLETIN  No.  190  [June, 

EXPEEIMENT  I:  NITRITE  BACTERIA  AND  PURE  ROCK 

PHOSPHATE 

The  purpose  of  this  experiment  was  to  test  the  ability  of  nitrite 
bacteria  to  dissolve  pure  rock  phosphate.  The  methods  of  determining 
their  ability  and  its  extent  consisted  in  actually  measuring  by  chem- 
ical analysis  the  amount  of  nitrogen  which  they  had  changed  from 
ammonia  to  nitrite  and  the  amount  of  phosphorus  and  calcium  which 
had  at  the  same  time  been  made  soluble. 

The  plan  of  the  experiment,  briefly  stated,  was  as  follows :  A  thin 
layer  (about  %  inch  thick)  of  a  nutrient  salt  solution  was  placed  in 
a  cone-shaped  glass  flask  of  about  one  quart  capacity  and  about  5 
inches  in  diameter  at  the  bottom.  In  this  solution  was  placed  a  definite 
amount  of  the  insoluble  pure  tricalcium  phosphate  and  a  definite 
amount  of  ammonium  salt.  The  flasks  and  materials  were  sterilized 
to  kill  all  bacteria  and  molds.  Nitrite  bacteria  isolated  from  corn- 
belt  soil  were  then  introduced  from  pure  cultures  or  directly  from  the 
soil.  -Cotton  plugs  were  kept  in  the  mouths  of  the  flasks  to  prevent 
the  entrance  of  other  bacteria.  The  flasks  were  kept  at  a  temperature 
of  82°  Fahrenheit.  Many  such  flasks  were  prepared,  and  later,  usually 
at  intervals  of  one  week,  the  contents  of  two  or  more  flasks  were 
analyzed  for  nitrogen  changed  or  oxidized  and  for  water-soluble  phos- 
phorus and  calcium.1 

JThe  details  of  the  experiment  are  given,  as  follows,  for  those  desiring  more 
information : 

The  flasks  were  one-liter  Erlenmeyers,  and  25  cc.  of  the  salt  solution  was 
added  to  each  one.  The  salt  solution  contained,  per  liter,  1  gram  sodium  chlorid, 
250  milligrams  potassium  sulfate,  100  milligrams  magnesium  sulfate,  and  3  drops 
ferric  chlorid. 

The  flasks  containing  the  nutrient  solution  were  plugged  with  cotton  and 
sterilized  in  the  autoclave.  The  salt  solution,  tricalcium  phosphate,  and  nitrogen 
solutions  were  analyzed  before  being  used  and  were  sterilized  before  being  added 
to  the  flasks.  These  chemicals  were  especially  prepared  to  free  them  from  im- 
purities. To  each  flask  was  added  45  milligrams  tricalcium  phosphate  and  10.585 
milligrams  nitrogen  as  ammonium  sulfate.  In  some  cases  the  nitrogen  was  in- 
creased to  21.17,  42.34,  and  84.68  milligrams  per  flask  to  test  the  effect  of  con- 
centration. Pure  cultures  of  nitrite  bacteria  were  used  in  some  experiments.  These 
had  been  isolated  on  silica  jelly  from  typical  corn-belt  soil.  In  some  experiments 
the  bacteria  were  added  directly  from  the  soil  in  5  cc.  of  an  infusion  made  of  2 
parts  water  and  1  part  soil.  The  soil  infusion  was  allowed  to  settle,  the  super- 
natant liquid  was  then  drawn  off  into  a  beaker  and  further  settling  allowed  be- 
fore the  infusion  was  added  to  the  flasks.  This  prevented  the  addition  of  soil 
particles,  which  might  furnish  a  free  base.  The  soil  infusion  appears  more  satis- 
factory than  pure  cultures. 

The  colonies  grew  on  the  surface  of  the  liquid,  forming  a  bluish  mass,  some 
developing  to  ^4  inch  in  diameter.  Typical  colonies  of  this  kind  were  isolated 
from  the  impure  soil  infusion  cultures  on  silica-jelly  plates.  Colonies  %  inch  in 
diameter  developed.  They  were  colorless  to  opalescent  at  first  and  later  a  glassy 
blue,  center  showing  yellow  after  fourteen  days  and  later  orange  yellow  to 
brown.  When  stained  with  gentian  violet,  they  appeared  as  typical  Nitrosomonas. 
Visible  growth  in  solution  was  slow  for  the  first  forty  days,  but  after  that  time  a 
very  profuse  surface  growth  developed  showing  large  blue  colonies  some  of  which 
were  drawn  up  the  sides  of  the  flask  by  the  surface  tension  of  the  liquid  and 


1916} 


SOIL  BACTERIA  AND  PHOSPHATES 


401 


In  Table  1  is  shown  the  relative  amounts,  by  weight,  of  nitrogen 
from  ammonium  sulfate  oxidized  to  nitrite  by  nitrite  bacteria  and  the 
amounts  of  phosphorus  and  calcium  made  soluble.  Each  figure  repre- 
sents the  average  of  duplicate  determinations. 

TABLE  1. — NITROGEN  OXIDIZED,  AND  PHOSPHORUS  AND  CALCIUM  MADE  SOLUBLE  BY 

NITRITE  BACTERIA* 
(Expressed  in  milligrams) 


Flask 

Duration 

Nitrogen 

Phosphorus 

Calcium 

No. 

in  days 

oxidized 

made  soluble 

made  soluble 

1 

28 

2.54 

4.08 

3.87    . 

2 

41 

3.81 

5.08 

5.60 

3 

41 

5.99 

8.40 

4 

48 

5.52 

9.56 

14.80 

5 

48 

4.88 

10.20 

18.40 

6 

55 

6.40 

12.85 

22.00 

7 

55 

6.40 

10.24 

23.52 

8 

62 

6.88 

16.00 

31.04 

9 

48 

3.61 

7.52 

13.60 

10 

62 

3.87 

8.76 

16.48 

11 

62 

5.84 

9.82 

16.00 

12 

62 

5.68 

11.28 

20.80 

13 

69 

6.03 

11.14 

22.40 

14» 

48 

5.76 

13.04 

24.80 

15' 

69 

4.60 

11.60 

19.20 

16 

139 

18.84 

41.56 

75.26 

*The  results  show  that  the  rate  of  solution  varies  even  under  conditions  made 
as  nearly  alike  as  possible.  Factors  tending  to  produce  these  variations  may  in- 
clude variations  in  the  number  of  bacteria  originally  introduced  and  in  the  diffusion 
of  carbon  dioxid  and  oxygen  thru  the  cotton  plugs. 

'Ammonium  nitrate  was  used  in  Flasks  14  and  15  in  place  of  ammonium  sul- 
fate. 

EXPLANATION  OF  RESULTS 

The  results  reported  in  Table  1  demonstrate  conclusively  that  phos- 
phorus and  calcium  are  made  soluble  while  the  nitrite  bacteria  oxi- 
dize ammonia  nitrogen  to  nitrite  nitrogen.  It  is  also  evident  that  the 
solubility  increases  with  increasing  time  of  action  of  the  bacteria. 

there  developed  to  a  large  size  (}4  inch  in  diameter).  Five  cubic  centimeters 
sterile  water  was  added  every  week  to  make  up  for  evaporation.  When  the  deter- 
minations were  made,  duplicate  flasks  were  filtered  thru  the  same  filter  (S.  and  S. 
blue  ribbon  589)  and  washed  with  25  cc.  nitrogen- free  cold  distilled  water.  Some- 
times double  filters  were  used.  The  whole  was  made  up  to  200  cc.  and  the  nitro- 
gen oxidized  determined  by  the  Devarda  method,  phosphorus  by  the  volumetric 
method,  and  calcium  by  the  permanganate  method. 

A  factor  which  may  account  for  deviations  in  the  results  was  the  slime  growth, 
which  by  absorption  or  by  clogging  of  the  filter  possibly  prevented  the  filtration  and 
washing  out  of  all  the  soluble  calcium  and  phosphorus.  Sterile  checks  were  in- 
cluded to  test  the  solubility  of  the  tricalcium  phosphate  in  pure  water,  in  the  salt 
solution,  in  the  presence  of  the  soil  infusion,  and  in  the  presence  of  the  ammonium 
salts,  singly  and  in  combination.  The  soluble  contents  of  the  flasks  developed  an 
acid  reaction,  as  required  by  the  formation  of  acid  phosphate  in  the  solution. 

Ten  cubic  centimeters  of  Flask  16  required  3.35  cc.  of  N/12.5  NaOH  with 
phenolphthalein  as  the  indicator  for  the  second  hydrogen  atom.  The  normality  of 
the  solution  was  found  to  be  N/37.2.  A  soil  with  20  percent  moisture  would  re- 
quire 535.9  pounds  of  calcium  carbonate  per  acre  to  neutralize  the  acid  produced 
by  the  bacteria. 


402  BULLETIN  No.  190  [June, 

An  inspection  of  the  figures  shows  that  there  is,  by  weight,  approxi- 
mately twice  as  much  phosphorus  and  four  times  as  much  calcium 
made  soluble  as  there  is  nitrogen  oxidized  by  the  bacteria.  As  an  aver- 
age of  the  results  from  thirteen  flasks  (Nos.  4  to  16),  we  find  that  from 
the  oxidation  of  56  pounds  of  nitrogen  115  pounds  of  phosphorus  and 
211  pounds  of  calcium  are  made  soluble.  The  results  from  Flasks 
1,  2,  and  3  are  not  included  in  the  ratio  calculated  or  discussed,  as 
there  appears  to  be  a  utilization  by  the  bacteria  themselves  of  the  cal- 
cium first  liberated,  the  bacteria  possibly  storing  it  up  in  their  bodies 
in  order  to  have  a  reserve  supply.  (Thru  an  error,  the  nitrogen 
oxidized  in  Flask  3  was  not  secured.) 

For  a  proper  understanding  of  the  results,  an  explanation  of  the 
ratio  of  oxidized  nitrogen  to  soluble  phosphorus  and  soluble  calcium 
is  essential. 

Ammonium  sulfate,  (NH4)2S04,  is  made  up  of  two  chemical  oppo- 
sites,  the  ammonium  (NH4)  being  alkaline  and  the  sulfate  (S04)  being 
an  acid  radicle.  When  they  unite,  two  ammonium  groups  are  required 
to  neutralize  one  sulfate  radicle. 

When  the  nitrite  bacteria  oxidize  the  ammonium  groups  (NH4) 
to  the  acid  radicle  (N02),  an  equivalent  amount  of  sulfate  radicle 
(S04)  is  free  to  act,  with  the  nitrous  acid  formed,  on  the  raw  rock 
phosphate.  As  much  calcium  is  required  to  combine  with  the  sulfate 
as  with  the  nitrite.  All  other  substances  which  might  combine  with 
the  acid  formed  or  freed  are  already  held  in  neutral  combination. 
Thus,  the  nitrogen  oxidized  unites  with  one-half  the  calcium  made 
soluble,  while  the  sulfate  radicle  (S04),  or  the  nitrate  radicle  (N03) 
(Flasks  14  and  15),  unites  with  the  other  half.  The  equations  below 
show  this  fact  for  ammonium  sulfate : 

2(NH4)2SO4+12O=4HNO2+  2H2SO4+4H2O 
4HNO2+Ca3(PO4)2=CaH4(PO4)2-{-2Ca(NO2), 
2H2SO4+Cas  (P04)  f=  CaH4  (PO4)  2-f  2CaSO4 

According  to  these  equations,  when  56  pounds  of  nitrogen  are 
changed  from  the  ammonia  form  to  the  nitrite  form,  with  both  the 
nitrous  acid  (HN02)  and  the  associated  sulfuric  acid  (H2S04)  acting 
on  the  pure  rock  phosphate,  124  pounds  of  phosphorus  and  240  pounds 
of  calcium  are  made  soluble  and  thus  occurs  the  double  action  obtained 
on  the  insoluble  phosphate  in  the  results  reported.  The  average  re- 
sults from  the  thirteen  duplicate  trials  show  that  115  parts  of  phos- 
phorus (instead  of  124)  and  211  parts  of  calcium  (instead  of  240) 
were  actually  found  in  solution,  and,  in  individual  cases  (Flasks  5,  10, 
14,  8,  9,  16),  the  experimental  results  approached  even  more  closely  to 
the  theoretical  amounts.  The  results  obtained  with  the  ammonium 
nitrate  are  in  agreement  with  those  obtained  with  ammonium  sulfate. 

Interpreted  in  terms  of  farm  practice,  these  results  mean  that 
every  pound  of  organic  nitrogen  in  manure  or  clover  residues  which  is 
converted  into  nitrate  may  make  slightly  more  than  one  pound  of 


1916]  SOIL  BACTERIA  AND  PHOSPHATES  403 

phosphorus  and  two  pounds  of  calcium  soluble  from  raw  rock  phos- 
phate, assuming  of  course  an  intimate  contact  of  phosphate  and  decay- 
ing organic  matter  and  that  all  the  nitrous  acid  acts  on  the  insoluble 
phosphate.  The  exact  proportions  are  56  of  nitrogen,  62  of  phosphorus, 
and  120  of  calcium,  as  is  readily  computed  from  the  reaction  between 
four  molecules  of  nitrous  acid  and  one  of  tricalcium  phosphate. 

When  insoluble  tricalcium  phosphate  (pure  rock  phosphate)  is 
converted  into  soluble  monocalcium  phosphate,  the  solution  is  made 
acid  on  account  of  the  increased  number  of  hydrogen  atoms  in  solu- 
tion., The  soluble  contents  of  the  flasks  used  in  this  experiment  should, 
therefore,  be  acid.  The  filtrates  were  tested  with  blue  litmus  paper 
and  found  to  be  acid.  Some  of  the  solutions,  especially  that  in  Flask  8, 
\vhich  represented  complete  solution  of  the  phosphate,  instantly  turned 
blue  litmus  red. 

The  bacteria,  after  dissolving  the  phosphate,  by  subsequent  action 
on  the  solution  make  some  of  the  phosphorus  insoluble  again,  possi- 
bly by  utilizing  it  for  their  own  growth.  This  actually  occurred  when 
the  filtrates  were  allowed  to  stand  a  long  time,  unless  sterilized.  It  is 
therefore  important  to  make  determinations  periodically,  as  a  rever- 
sion of  the  phosphate  or  a  precipitation  of  calcium  sulfate  from  a  con- 
centration of  the  solution  by  evaporation  of  the  water,  may  obscure  the 
true  ratios. 

EXPERIMENT  II:  NITRATE  BACTERIA  AND  PURE  ROCK 

PHOSPHATE 

The  results  of  an  experiment  to  test  the  effect  of  the  nitrate  bac- 
teria on  pure  tricalcium  phosphate  are  seen  by  reference  to  Table  2. 
While  all  the  nitrogen  in  one  case  and  nearly  all  in  the  other  had  been 
oxidized  to  nitrate,  practically  no  soluble  phosphorus  or  calcium  was 
found  above  that  in  the  check ;  which  fact  of  course  supports  the  theory 
that  no  solution  of  phosphate  is  to  be  expected  by  the  action  of  nitrate 
bacteria. 

TABLE  2. — NITROGEN  OXIDIZED  FROM  SODIUM  NITRITE  TO  NITRATE,  AND  PHOSPHORUS 

AND  CALCIUM  MADE  SOLUBLE  BY  NITRATE  BACTERIA 

(Expressed  in  milligrams) 


Flask 
No. 

Duration 
in  days 

Nitrogen 
oxidized 

Phosphorus 
soluble 

Calcium 
soluble 

1 

2 

41 
62 

21.45 
18.67 

.00 
.00 

.09 
.00 

SOLUBILITY  OF  PHOSPHATES  BY  CARBON  DIOXID1 

Carbon  dioxid  (€02)  is  produced  in  large  amounts  in  soils  by  bac- 
terial action  on  proteins,  sugars,  starches,  fiber,  and  other  compounds. 

*Carbon  dioxid  is  a  gas,  but  when  dissolved  in  water,  it  forms  carbonic  acid, 
or  carbonated  water. 


404  BULLETIN  No.  190  [June, 

It  is  exhaled  by  bacteria  in  respiration  just  as  it  is  by  man.  It  has 
been  found  that  one  pound  of  non-symbiotic  nitrogen-fixing  bacteria 
(Azotobacter)  exhaled  1.27  pounds  of  carbon  dioxid  in  24  hours.  A 
man  weighing  150  pounds  and  doing  hard  muscular  work  exhales  only 
3.74  pounds  of  carbon  dioxid  in  24  hours. 

The  enormous  production  of  carbon  dioxid  by  soil  bacteria  is  due 
to  their  feeding  upon  easily  oxidizable  carbon  and  to  their  rapid  mul- 
tiplication. It  is  well  known  that  manure  and  crop  residues  added  to 
the  soil  greatly  increase  the  carbon  dioxid  produced,  as  these  materials 
are  good  food  for  bacteria.  In  one  case  a  normal  application  of  manure 
increased  the  carbon  dioxid  produced  33  percent.  Very  little  carbon 
is  retained  by  the  bacteria  compared  to  that  given  off. 

The  carbon  dioxid  produced  by  bacteria  is  absorbed  in  the  soil 
moisture  and  when  in  contact  with  raw  rock  phosphate  will  dissolve 
it  according  to  the  following  equation : 

4H2COJ+Ca3(P04)2=2Ca(HCO,)2+CaH1(P04)1 

According  to  this,  176  pounds  of  carbon  dioxid  make  62  pounds  of 
phosphorus  soluble. 

It  is  a  simple  matter  to  show  the  solubility  of  raw  rock  phosphate 
in  water  saturated  with  carbon  dioxid.  This  was  accomplished  as 
early  as  1868,  by  Knop.  However,  when  soil  is  treated  with  carbon- 
ated water  for  long  periods,  only  relatively  small  amounts  of  the  phos- 
phorus present  can  be  found  in  a  soluble  form;  and  when  bacteria 
which  produce  carbon  dioxid  in  large  amounts  are  allowed  to  act  in 
soils,  not  much  soluble  phosphorus  is  produced  unless  large  applica- 
tions of  carbonaceous  material  have  been  made  for  the  bacteria  to  feed 
upon.  When  this  is  done,  a  more  appreciable  solubility  is  found. 

Conditions  are  seldom  favorable  for  more  than  a  small  portion  of 
the  carbon  dioxid  produced  in  the  soil  to  act  in  the  liberation  of  phos- 
phorus from  raw  rock  phosphate.  Much  of  the  carbon  dioxid  is  dissi- 
pated in  other  ways  and  the  greater  portion  returns  to  the  air,  it  be- 
ing a  very  volatile  compound.  This  is  evident  from  the  work  of  Kro- 
ber,  who  found  under  very  suitable  conditions  that  it  required  the 
production  of  122  pounds  of  carbon  dioxid  to  liberate  one  pound  of 
soluble  phosphorus  from  pure  rock  phosphate. 

It  has  long  been  known  that  plants  excrete  carbon  dioxid  from  their 
roots.  Kossowitsch  found  that  the  roots  of  mustard  plants  produced 
large  amounts  of  carbon  dioxid  during  a  growing  season.  Stocklasa 
studied  the  production  of  carbon  dioxid  from  wheat  and  clover  roots 
and  found  that  the  daily  production  was  much  greater  with  the  clover 
than  with  the  wheat. 

The  effectiveness  of  carbon  dioxid  excreted  by  plant  roots  depends 
upon  the  number  of  plants  per  acre,  the  kind  of  plant,  and  the  kind 
and  amount  of  phosphate  applied.  The  larger  the  application  the 
greater  the  contact  with  the  plant  roots.  It  has  been  demonstrated 
by  investigations  with  pot  cultures  at  the  University  of  Illinois  that 


1916]  SOIL  BACTERIA  AND  PHOSPHATES  405 

common  farm  crops  possess  some  ability  to  utilize  rock  phosphate  with- 
out organic  matter  and  that  their  ability  to  do  this  is  increased  by  in- 
creased applications  of  the  rock  phosphate.  It  has  also  been  shown 
that  cereals  possess  this  ability  as  well  as  legumes. 

Rain  water  is  another  source  of  carbon  dioxid,  becoming  charged 
with  the  gas  from  the  air,  in  which  there  are  four  parts  in  10,000 ;  and 
such  water  possesses  power  to  dissolve  minerals,  such  as  rock  phos- 
phate. That  carbon  dioxid  is  an  important  solvent  of  minerals  is  well 
understood. 

SOLUBILITY  OF  PHOSPHATES  BY  ORGANIC  ACIDS 

The  acid  of  vinegar  (acetic),  of  rancid  butter  (butyric),  of  sour 
milk  (lactic),  and  many  other  similar  acids  result  from  bacterial  and 
mold  action  on  carbohydrates,  proteins,  and  fats  in  the  soil.  These 
acids  may  act  on  phosphates  and  render  them  soluble.  They  are  much 
stronger  in  their  action  than  carbonic  acid  and  weaker  than  nitrous 
or  nitric  acid.  They  are  produced  in  very  small  amounts  compared 
with  the  production  of  carbon  dioxid,  but  perhaps  in  larger  amounts 
than  nitrous  acid.  The  percentage  of  phosphorus  made  soluble  by 
these  acids  is  probably  higher  under  similar  conditions  than  that  made 
soluble  by  carbonic  acid. 

Sackett,  Patten,  and  Brown  of  the  Michigan  Agricultural  Experi- 
ment Station,  and  Koch  and  Krober  of  Germany  have  shown  that 
soil  bacteria  which  produce  organic  acids  make  large  amounts  of  bone 
meal  and  raw  rock  phosphate  soluble.  The  addition  of  limestone  with 
the  insoluble  phosphates  prevents  the  detection  of  soluble  phosphates. 
The  undesirable  condition  created  by  the  intimate  contact  of  ground - 
limestone  and  rock  phosphate  in  the  soil  is  largely  avoided  by  the  meth- 
ods of  application  recommended  by  the  Illinois  Station. 

IMPORTANCE  AND  EXTENT  OF  THE  ACTION  OF  NITRITE 

BACTERIA 

It  has  already  been  shown  that  the  nitrite  bacteria  make  phos- 
phorus and  calcium  soluble  from  pure  rock  phosphate  and  that  the 
action  conforms  to  a  definite  chemical  ratio.1 

The  nitrous  acid2  produced  may  act  upon  compounds  of  iron, 
aluminum,  potassium,  sodium,  or  magnesium  which  occur  in  soils,  or 
it  may  act  upon  tricalcium  phosphate,  calcium  silicate,  or  calcium  car- 
bonate, if  present.  For  this  reason,  it  has  been  recommended  that  the 
ideal  practice  to  obtain  the  greatest  solubility  of  the  raw  rock  phos- 
phate is  to  turn  it  under  in  intimate  contact  with  organic  matter,  and, 

JIt  was  found  that  the  action  of  the  nitrite  bacteria  was  the  same  on  the 
natural  raw  rock  phosphate  as  on  the  pure  rock  phosphate,  but  more  extensive 
experiments  with  the  natural  rocks  will  be  reported  later. 

-It  is  not  necessary  to  assume  that  the  nitrous  acid  produced  by  the  bacteria 
accumulates  in  the  soil  to  a  noticeable  extent. 


406 


BULLETIN  No.  190 


[June, 


if  needed,  to  apply  ground  limestone  after  plowing  or  at  some  other 
point  in  the  crop  rotation. 

The  role  the  plant  plays  in  this  process  of  utilizing  natural  plant 
food  materials  is  highly  important :  it  acts  as  a  pump,  removing  from 
the  soil  the  soluble  phosphorus,  calcium,  and  nitrogen,  as  they  are 
formed,  thus  giving  room  for  more  soluble  phosphorus,  calcium, 
and  nitrogen  to  be  formed.  Under  these  conditions  the  bacteria  are 
stimulated  to  put  forth  their  best  efforts.  When  the  soluble  nitrogen, 
phosphorus,  and  calcium  accumulate,  they  probably  tend  to  reduce  the 
activity  of  the  nitrite  bacteria,  because  bacteria  do  not  thrive,  as  a 
rule,  in  an  excess  of  their  own  product,  but  fortunately  their  action 
never  ceases  while  suitable  conditions  exist. 

In  Table  3  are  presented  the  actual  amounts  of  phosphorus,  calcium, 
and  nitrogen  required  by  standard  crops,  and  the  amounts  of  phos- 
phorus and  calcium  which  would  be  made  soluble  if  all  the  nitrogen 
required  for  the  crop  should  be  oxidized  to  nitrate  and  should  act 
upon  pure  rock  phosphate. 

TABLE  3. — PHOSPHORUS,  CALCIUM,  AND  NITROGEN  EEQUIRED  BY  CROPS,  COMPARED 
WITH  THAT  POSSIBLE  OF  SOLUTION  WHEN  NITRITE  BACTERIA  ACT  UPON  TRI- 
CALCIUM  PHOSPHATE 

(Expressed  in  pounds) 


Crop 

Nitrogen 

Phosphorus 

Calcium 

Eequired 

Required]  Possible 

Eequired  |  Possible 

Corn 
Grain,  100  bu  

150 
96 

97 
76 

23 
16 

16 
9 

166 
107 

108 

84 

22 
11 

17 

20 

321 
206 

208 
163 

Stover,  3  tons   

Cobs,  %  ton  

Wheat 
Grain,  50  bu  

Straw,  2%  tons  

Oats 
Grain,  100  bu  

Straw,  2V>  tons  

Timothy,  3  tons  

The  figures  show  that  there  is  possible  of  solution  from  this  bio- 
chemical process  about  7  times  as  much  phosphorus  as  corn,  wheat,  or 
oats  require,  and  9  times  as  much  as  timothy  requires.  Greater  dif- 
ferences occur  in  the  calcium  figures,  there  being  possible  of  solution 
]4  times  that  required  for  corn,  18  times  that  required  for  wheat,  12 
times  that  required  for  oats,  and  8  times  that  required  for  timothy.  It 
is  evident  that  nitrite  bacteria  find  in  pure  rock  phosphate  a  highly 
satisfactory  source  of  phosphorus. 

The  question,  Will  the  calcium  of  raw  rock  phosphate  suffice  to 
neutralize  the  acid  produced  by  nitrite  bacteria ;  and,  if  so,  will  phos' 
phorus  be  made  soluble  ?  has  been  answered  in  the  affirmative  from  the 
results  of  this  work. 

—65 


UNIVERSITY  OF  ILLINOIS-URBANA 


